The primary objective of this proposal is to determine the physiological and pharmacological role and mechanisms of adenosine and adenine nucleotide regulation of adenyl cyclase in mammalian cells. We will attempt to purify the adenosine sensitive adenyl cyclase with purification of the plasma membrane. Highly purified plasma membrane vesicles will be formed using procedures originally developed for the formation of plasma membrane vesicles by spontaneous vesiculation of erythrocytes ghosts. Various chemical factors affecting the formation of plasma membrane vesicles and the stabilization of the adenosine and hormone sensitive cyclase will be evaluated. The purified adenosine-sensitive adenyl cyclase will be used to determine the mechanism of methylxanthine inhibition and the relationship of the adenosine site of action to the receptors of sites for catecholamine, fluoride, PGE1 and Gpp(NH)p. Attempts will be made to characterize the adenosine receptor by binding studies with adenosine, adenosine analogues and competitive inhibitors of adenosine action such as the methylxanthines. Adenyl cyclase from other cell lines which respond to adenosine with increased levels of cyclic AMP such as the Chinese hamster ovary line and human lung fibroblast lines will be also be examined as reference systems to determine if the mechanism of adenosine action is similar to that in the 132-1N1 line. Another objective is to determine if the pharmacologically important vasodilator effects of adenosine on coronary artery smooth muscle are mediated by increased or decreased cyclic AMP, and if so, if the mechanism of adenosine stimulation is similar to its action on the cultured lines, i.e. to regulate adenyl cyclase. We will attempt to evaluate whether any of the actions of methylxanthines, dypyridamole, and/or other structurally related drugs with important cardiovascular effects are related to an inhibition of adenosine action. We will continue to determine the universality of adenosine action on cyclic AMP metabolism in other cultured cell lines and tissue preparations.